Fermentation process for the production of β-carboline derivatives by Myrothecium verrucaria

ABSTRACT

A process for the production of β-carboline derivatives of general formula I ##STR1## in which X represents a hydrogen atom or a halogen atom, 
     Y represents a carbon-oxygen bond or a methyl group, 
     R 1  represents an alkyl group with up to 6 carbon atoms and 
     R 2  represents an alkyl group with up to 4 carbon atoms, 
     from 1,2,3,4-tetrahydro-β-carboline derivatives of general formula II ##STR2## in which X, Y, R 1  and R 2  have the above-mentioned meaning, is described, which is characterized in that the 1,2,3,4-tetrahydro-β-carboline derivatives are fermented with a fungi culture of genus Fusarium or Myrothecium.

The invention relates to a process for the production of β-carbolinederivatives of general formula I ##STR3## in which X represents ahydrogen atom or a halogen atom,

Y represents a carbon-oxygen bond or a methyl group,

R₁ represents an alkyl group with up to 6 carbon atoms and

R₂ represents an alkyl group with up to 4 carbon atoms,

from 1,2,3,4-tetrahydro-β-carboline derivatives of general formula II##STR4## in which

X, Y, R₁ and R₂ have the above-mentioned meaning.

β-carboline derivatives of general formula I are, as is generally known,pharmacologically effective substances, which can be produced, forexample, by chemical dehydrogenation of 1,2,3,4-tetrahydro-β-carbolinederivatives of general formula II (EP-A 0130140, EP-A 0234173 and EP-A0239669). But this chemical dehydrogenation is quite expensive and theyields that can be achieved in this way are low.

It has now been found that the dehydrogenation of1,2,3,4-tetrahydro-β-carboline derivatives of general formula II can beperformed in a simple way under nonpolluting conditions and whileachieving satisfactory yields, by these compounds being fermented with afungal culture of genera Fusarium or Myrothecium. According to thestudies so far available, fungal cultures of the species Myrotheciumverrucaria seem to be especially suitable for the performance of theprocess according to the invention.

It is very surprising for one skilled in the art that the1,2,3,4-tetrahydro-β-carboline derivatives of general formula II can beconverted with the help of the process according to the invention intoβ-carboline derivatives of the general formula I since suchdehydrogenation on heteroaromatic compounds has not been previouslydescribed. The fact that the ester groups of the substrates are notcleaved in this fermentation is also surprising.

The process according to the invention is performed under the samefermentation conditions, which are also used in the knownmicrobiological conversions of substrates with fungal cultures.

Under the culture conditions usually used for fungal cultures, submergedcultures are cultivated in a suitable nutrient medium with aeration.Then, the substrate (dissolved in a suitable solvent or in emulsifiedform) is added to the cultures and fermented until a maximum substrateconversion is achieved.

Suitable substrate solvents are, for example, methanol, ethanol, glycolmonomethyl ether, dimethylformamide or dimethyl sulfoxide. Theemulsification of the substrate can be achieved, for example, by thelatter being injected in micronized form or dissolved in awater-miscible solvent (such as methanol, ethanol, acetone, glycolmonomethyl ether, dimethylformamide or dimethyl sulfoxide) under strongturbulence in (preferably decalcified) water, which contains the usualemulsification aids. Suitable emulsification aids are nonionogenicemulsifiers, such as, for example, ethylenoxy adducts or fatty acidesters of polyglycols. As suitable emulsifiers, the commerciallyavailable wetting agents Tegin®, Tween® and Span® can be mentioned asexamples.

The optimum substrate concentration, substrate adding time andfermentation period depend on the type of substrate and microorganismand fermentation conditions used. These values, as is generallynecessary in microbiological steroid conversions, have to be determinedin individual cases by preliminary tests, as they are familiar to oneskilled in the art.

The 1,2,3,4-tetrahydro-β-carboline derivatives of general formula IIused as initial material for the process according to the invention canhave as substituent X, for example, a hydrogen atom or a chlorine atompreferably present in p-position. Suitable substituents R₁ of thesubstrate are, for example, the methyl group, the ethyl group, thepropyl group, the isopropyl group or the tert-butyl group. Assubstituent R₂ of the substrates, for example, the ethyl group, thepropyl group, the isopropyl group and in particular the methyl group canbe mentioned. These compounds are known, or can be produced analogouslyto the process which is described in EP-A 0130140.

The following embodiment is used for a more detailed explanation of theprocess according to the invention.

EXAMPLE

a) A 2 1 Erlenmeyer flask with 500 ml of sterile nutrient mediumcontaining

    ______________________________________                                        3                % glucose                                                    1                % cornsteep liquor                                           0.2              % NaNO.sub.3                                                 0.1              % KH.sub.2 PO.sub.4                                          0.05             % KCl                                                        0.002            % FeSO.sub.4                                                 0.2              % K.sub.2 HPO.sub.4                                          ______________________________________                                    

with a pH of 6.1 is inoculated with 5 ml of a suspension of aMyrothecium verrucaria DSM 2087 culture and shaken for 48 hours at 30°C. with 180 rpm.

b) 20 Erlenmeyer flasks (500 ml) with 100 ml of sterile nutrient mediumeach containing

1.0 % cornsteep liquor

1.25 % soybean powder

adjusted to pH 6.2

are inoculated with 10 ml of Myrothecium verrucaria growing culture eachand incubated for 7 hours on a rotary shaker with 180 rpm at 30° C.

Then, 0.04 g of6-benzyloxy-4-methoxymethyl-1,2,3,4-tetrahydro-β-carboline-isopropylester dissolved in 1 ml of dimethylformamide and sterilized byfiltration is added to each culture and fermented for another 113 hours.

c) The combined cultures are extracted with methyl isobutyl ketone andthe extract is concentrated by evaporation under vacuum in a rotaryevaporator at a maximum of 50° C. Then, a purification by chromatographyon a silica gel column is performed.

0.5 g of 6-benzyloxy-4-methoxymethyl-β-carboline-3-carboxylic acidisopropyl ester of melting point 150°-151° C. is obtained.

We claim:
 1. A process for the production of β-carboline derivatives of the formula I ##STR5## in which X represents a hydrogen atom or a halogen atom,Y represents a carbon-oxygen bond or a methylene group, R₁ represents an alkyl group of 1 to 6 carbon atoms and R₂ represents an alkyl group of 1 to 4 carbon atoms,from 1,2,3,4-tetrahydro-β-carboline derivatives of the formula II ##STR6## in which X, Y, R₁ and R₂ have the above-mentioned meaning, comprising culturing Myrothecium verrucaria in a nutrient medium containing the 1,2,3,4-tetrahydro-β-carboline derivatives under conditions effective to produce the β-carboline derivatives of formula I, and recovering the β-carboline derivatives produced. 